Major depressive disorder (MDD) is characterized by reductions in the density and size of neuronal and glial cells in prefrontal cortex. We find these cellular changes to be age-dependent as younger depressed show prominent reductions in the density of glial cells (astrocytes), whereas older depressed have marked reductions in the density of pyramidal (presumably glutamate) neurons and calbindin-immunoreactive interneurons (mostly co-localizing GABA). Since, astrocytes regulate concentration of glutamate, an excess of which is neurotoxic, we propose that an early deficit in astrocytes in MDD could lead to an increase in the extracellular concentration of glutamate and to a reduction in pyramidal and GABA neurons later in life. Hence, there may be imbalances in GABA/glutamate homeostasis that are consistent with neuroimaging studies revealing changes in levels of GABA and glutamate in MDD which are reversible with antidepressant (SSRI) treatment. Cortical neurons are regulated in complex ways by serotonin acting (at least) at serotonin- 1A and -2A receptors located on these neurons and astrocytes. Pathology in ascending serotoninergic axons and postsynaptic receptors may be related to the activity, number and size of glutamate and/or GABA neurons and astrocytes in MDD. To date, there have been no studies on the expression or localization of serotonin receptors on specific cortical cell types in depression. The overall hypothesis is that in depression there will be age-dependent reductions in the density of astrocytes, glutamate pyramidal neurons and GABA interneurons, and that the expression of regulatory serotonin-1A and -2A receptors on these cells will be altered. These cell reductions will also be correlated with an age-related loss of serotonin innervation in prefrontal layers. To test these hypotheses, we will directly identify and quantify the packing density of astrocytes and glutamate and GABA neurons expressing mRNA for specific proteins (Aim 1). Moreover, we will assess the integrity of the serotonin system regulators (postsynaptic receptors and presynaptic axons) of prefrontal cells by estimating the proportion of cell types expressing mRNA for serotonin-1 A and -2A receptors (Aim 2), and the density of serotonin axons expressing the serotonin transporter (Aim 3). Double in situ hybridization, immunohistochemistry and 3-D cell counting techniques will be used in the same postmortem tissue sampled from the prefrontal cortex of younger and older subjects with MDD and non-depressed controls as used in our cell counting studies. This project will identify the cellular substrates of glutamate, GABA and serotonin interactions in the cortex and their potential role in the etiology, pathophysiology and age-related progression of depression. It may also reveal novel targets for preventing depressive illness and better antidepressant drug treatment.